Terpenes are hydrocarbons that are commonly found in plants and seeds. Certain naturally occurring terpenes, such as the cyclic monoterpenes, may be used in, e.g., fuel compositions and solvents for art restoration. These monoterpenes include the monocyclic monoterpenes such as limonene and terpinolene and the bicyclic monoterpenes such as α-pinene, β-pinene, bornyl diphosphate, sabinene and camphene. It has been recently discovered that the tricyclic monoterpene, tricyclene which is distinct from the monocyclic and bicyclic monoterpenes delineated above has very good octane properties and oxidative stability and therefore may be better suited for use as a gasoline component than other monoterpenes. Tricyclene may be obtained from natural sources. It can, for example, be extracted from a plant source, such as Cordia cylindrostachya and Salvia fruiticosa. It may also be made synthetically, for example by the isomerization of α-pinene into tricyclene and camphene (C M Lopez et al., (2001) React Kinet Catal Lett 74:163-170).
In biological systems, terpenes are synthesized from products and intermediates of carbohydrate metabolism by numerous enzyme-catalyzed reactions in the mevalonate (MEV) pathway or 1-deoxy-D-xylulose-5-phosphate (DXP) pathway and are assembled with five-carbon building blocks called isoprene units through the conversion of isopentenyl pyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP). See FIG. 1 and P. Dewitt (2002) Nat. Prod. Rep., 19:181-222. A transferase is used to elongate the isoprene units to obtain the corresponding monoterpene precursor geranyl pyrophosphate (GPP). Terpene synthases (also sometimes referred to as terpene cyclases) catalyze formation of the monoterpene products by a series of carbocation intermediates that can undergo a variety of cyclizations.
Reports describe microbial strains that have been engineered to produce terpenes (V J J Martin et al., (2003) Nature Biotechnol 21:796-802 and Takahashi et al., (2007) Biotechnol. Bioeng. 97:170-181); however, the inventors are not aware of any reports of a strain that has been engineered to produce tricyclene. Mutants of a pinene synthase were reported to produce trace amounts of tricyclene in vitro from GPP (Hyatt and Croteau (2005) Arch. Biochem. Biophys. 439: 222-223). Likewise, a terpene synthase from Arabidopsis thaliana was reported to generate a small amount of tricyclene in vitro (J. Bohlmann et al., (2002) Arch. Biochem. Biophys. 375:261-269). However, when the inventors of the present invention expressed either of these pinene synthases or the A. thaliana terpene synthase in E. coli, together with the mevalonate pathway to supply GPP in vivo they found tricyclene was not produced.